Mass spectrometry is an important tool in the analysis of a wide range of chemical compounds. Specifically, mass spectrometers can be used to determine the molecular weight of sample compounds. The analysis of samples by mass spectrometry consists of three main steps—formation of gas phase ions from sample material, mass analysis of the ions to separate the ions from one another according to ion mass, and detection of the ions. A variety of means exist in the field of mass spectrometry to perform each of these three functions. The particular combination of means used in a given spectrometer determine the characteristics of that spectrometer.
To mass analyze ions, for example, one might use a magnetic (B) or electrostatic (E) analyzer. Ions passing through a magnetic or electrostatic field will follow a curved path. In a magnetic field the curvature of the path will be indicative of the momentum-to-charge ratio of the ion. In an electrostatic field, the curvature of the path will be indicative of the energy-to-charge ratio of the ion. If magnetic and electrostatic analyzers are used consecutively, then both the momentum-to-charge and energy-to-charge ratios of the ions will be known and the mass of the ion will thereby be determined. Other mass analyzers are the quadrupole (Q), the ion cyclotron resonance (ICR), the time-of-flight (TOF), and the quadrupole ion trap analyzers.
Before mass analysis can begin, however, gas phase ions must be formed from sample material. If the sample material is sufficiently volatile, ions may be formed by electron impact (EI) or chemical ionization (CI) of the gas phase sample molecules. For solid samples (e.g. semiconductors, or crystallized materials), ions can be formed by desorption and ionization of sample molecules by bombardment with high energy particles. Secondary ion mass spectrometry (SIMS), for example, uses keV ions to desorb and ionize sample material. In the SIMS process a large amount of energy is deposited in the analyte molecules. As a result, fragile molecules will be fragmented. This fragmentation is undesirable in that information regarding the original composition of the sample—e.g., the molecular weight of sample molecules—will be lost.
For more labile, fragile molecules, other ionization methods now exist. The plasma desorption (PD) technique was introduced by Macfarlane et al. in 1974 (Macfarlane, R. D.; Skowronski, R. P.; Torgerson, D. F., Biochem. Biophys. Res Commun. 60 (1974) 616). Macfarlane et al. discovered that the impact of high energy (MeV) ions on a surface, like SIMS would cause desorption and ionization of small analyte molecules, however, unlike SIMS, the PD process results also in the desorption of larger, more labile species—e.g., insulin and other protein molecules.
Lasers have been used in a similar manner to induce desorption of biological or other labile molecules. See, for example, Van Breeman, R. B.: Snow, M.: Cotter, R. J., Int. J. Mass Spectrom. Ion Phys. 49 (1983) 35; Tabet, J. C.; Cotter, R. J., Anal. Chem. 56 (1984) 1662; or Olthoff, J. K.; Lys, I.: Demirev, P.: Cotter, R. J., Anal. Instrument. 16 (1987) 93. Cotter et al. modified a CVC 2000 time-of-flight mass spectrometer for infrared laser desorption of involatile biomolecules, using a Tachisto (Needham, Mass.) model 215G pulsed carbon dioxide laser. The plasma or laser desorption and ionization of labile molecules relies on the deposition of little or no energy in the analyte molecules of interest. The use of lasers to desorb and ionize labile molecules intact was enhanced by the introduction of matrix assisted laser desorption ionization (MALDI) (Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshica, T., Rapid Commun. Mass Spectrom. 2 (1988) 151 and Karas, M.; Hillenkamp, F., Anal. Chem. 60 (1988) 2299). In the MALDI process, an analyte is dissolved in a solid, organic matrix. Laser light of a wavelength that is absorbed by the solid matrix but not by the analyte is used to excite the sample. Thus, the matrix is excited directly by the laser, and the excited matrix sublimes into the gas phase carrying with it the analyte molecules. The analyte molecules are then ionized by proton, electron, or cation transfer from the matrix molecules to the analyte molecules. This process, MALDI, is typically used in conjunction with time-of-flight mass spectrometry (TOFMS) and can be used to measure the molecular weights of proteins in excess of 100,000 daltons.
Atmospheric pressure ionization (API) includes a number of methods. Typically, analyte ions are produced from liquid solution at atmospheric pressure. One of the more widely used methods, known as electrospray ionization (ESI), was first suggested by Dole et al. (M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, M. B. Alice, J. Chem. Phys. 49, 2240, 1968). In the electrospray technique, analyte is dissolved in a liquid solution and sprayed from a needle. The spray is induced by the application of a potential difference between the needle and a counter electrode. The spray results in the formation of fine, charged droplets of solution containing analyte molecules. In the gas phase, the solvent evaporates leaving behind charged, gas phase, analyte ions. Very large ions can be formed in this way. Ions as large as 1 MDa have been detected by ESI in conjunction with mass spectrometry (ESMS).
ESMS was introduced by Yamashita and Fenn (M. Yamashita and J. B. Fenn, J. Phys. Chem. 88, 4671, 1984). To establish this combination of ESI and MS, ions had to be formed at atmospheric pressure, and then introduced into the vacuum system of a mass analyzer via a differentially pumped interface. The combination of ESI and MS afforded scientists the opportunity to mass analyze a wide range of samples. ESMS is now widely used primarily in the analysis of biomolecules (e.g. proteins) and complex organic molecules.
In the intervening years a number of means and methods useful to ESMS and API-MS have been developed. Specifically, much work has focused on sprayers and ionization chambers. In addition to the original electrospray technique, pneumatic assisted electrospray, dual electrospray, and nano electrospray are now also widely available. Pneumatic assisted electrospray (A. P. Bruins, T. R. Covey, and J. D. Henion, Anal. Chem. 59, 2642, 1987) uses nebulizing gas flowing past the tip of the spray needle to assist in the formation of droplets. The nebulization gas assists in the formation of the spray and thereby makes the operation of the ESI easier. Nano electrospray (M. S. Wilm, M. Mann, Int. J. Mass Spectrom. Ion Processes 136, 167, 1994) employs a much smaller diameter needle than the original electrospray. As a result the flow rate of sample to the tip is lower and the droplets in the spray are finer. However, the ion signal provided by nano electrospray in conjunction with MS is essentially the same as with the original electrospray. Nano electrospray is therefore much more sensitive with respect to the amount of material necessary to perform a given analysis.
Furthermore, High performance liquid chromatography (“HPLC”) in combination with mass spectrometry has become an important tool in the analysis of a wide range of chemical and biological samples. When using conventional HPLC the time typically required for the elution of a given sample component (i.e., the time from when it starts to come out of the column to when it finished coming out of the column) is typically a few seconds. However, the time required to mass analyze a compound is much shorter (0.1 seconds or less). When using TOF mass analysis, the time needed to produce a mass spectrum may be as little as 0.01 seconds. As a result, one may, in principle, analyze the effluent from a number of columns simultaneously.
For example, FIG. 1 depicts a method and apparatus for multiplexing four spray needles 12, 14, 16 & 18 in an electrospray ionization source according to Kassel et al. U.S. Pat. No. 6,066,848 (“Kassel”). As described in Kassel, effluent from four HPLC columns 2, 4, 6 & 8 is injected into spray needles 12, 14, 16 & 18. The electrospray and subsequent ions produced by sprayers 12, 14, 16 & 18 are accelerated towards plate 36 by a potential between spray needles 12, 14, 16 & 18 and plate 36 and plate 38. Plate 36 includes aperture 32 located in an off-center position. During use plate 36 is rotated about its center (as indicated by arrows 34) and aperture 32 is aligned sequentially with spray needle 12, spray needle 16, spray needle 18, and spray needle 14, in turn. Ions produced by the sprayer aligned with aperture 32 pass through aperture 32 and on to orifice 20. These ions pass through orifice 20 and into mass spectrometer 10. In mass spectrometer 10, the ions are analyzed to determine their mass and abundance. As disk 36 is rotated, ions from the different sprayers (and the different HPLC columns) are sampled and mass spectra are produced—one mass spectrum for each spray needle 12, 14, 16 & 18 per each rotation of disk 36. The mass spectra may then be labeled electronically so as to associate the mass spectra with the sprayer (and HPLC column) from which they originate.
As described in Kassel, plate 36 serves as a “blocking” device, which moves in order to block the sample spray from all but one of spray needles 12, 14, 16 & 18 at any given time. Such a method and apparatus for multiplexing sample sprays has disadvantages. First, sampling orifice 20 is maintained in a fixed position with respect to spray needles 12, 14, 16 & 18. In such an arrangement, optimum conditions cannot be satisfied for each individual sprayer position with respect to the sampling orifice. Rather, an optimum geometry between sampling orifice 20 and all sprayers as a whole is optimized. Second, because plate 36 merely serves as a “blocking” device, significant portions of the sample spray is wasted (or lost) during each analysis (i.e., any sample spray that is blocked by plate 36 and does not pass through aperture 32).
Other techniques to sample ions from multiple ion sprayers are also known. One such method, similar to Kassel, as shown in FIG. 2, is an eight-way multiplexed electrospray inlet as disclosed by Robert Bateman et al., “Multiple LC/MS: Parallel and Simultaneous Analyses of Liquid Streams by LC/TOF Mass Spectrometry Using a Novel Eight-Way Interface”, American Society for Mass Spectrometry, 1998 (“Bateman”). Bateman discloses sampling cone 66 surrounded by rotating cylinder 68 (e.g., in a manner shown by arrow 74) having apertures 64 & 65 and sprayers 42, 44, 46, 48, 50, 52, 54 and 56 evenly spaced in an arc around cylinder 68. When sprayed, the sample droplets travel through aperture 64 or 65 (i.e., depending on which aperture is positioned in front of the spraying sprayer) to sampling cone 66, which is at the center of cylinder 68. Unlike Kassel (FIG. 1), rather than using a blocking plate (or disk), Bateman teaches a rotating cylinder 68 having apertures (64 & 65) for allowing the sample spray to pass therethrough and into sampling cone 66. Sampling cone 66 then transfers ions from atmospheric pressure region of source block 40 into a vacuum system of mass spectrometer 70, as indicated by arrow 72. Again, as disclosed in Kassel, the method and apparatus disclosed by Bateman uses a “blocking” device to prevent unwanted sample from entering the mass analyzer at a given point in time.
Also, methods for sampling solutions from different sprayers without using a multiplexing technique are known. For example, FIG. 3 depicts a multi-ESI-sprayer, multi-nozzle time-of-flight mass spectrometer as disclosed in Longfei Jiang and Mehdi Moini, “Development of Multi-ESI-Sprayer, Multi-Atmospheric-Pressure-inlet Mass Spectrometry and Its Application to Accurate Mass Measurement Using Time-of-Flight Mass Spectrometry”, Anal. Chem. 72,20 (2000) (“Jiang”). An elevated pressure ion source always has an ion production region (wherein ions are produced) and an ion transfer region (wherein ions are transferred through differential pumping stages and into the mass analyzer). Typically, the ion production region is at an elevated pressure—most often atmospheric pressure—with respect to the analyzer. Disclosed in Jiang is the use of a multitude of sprayers 14 with two differential pumping stages 90 & 96. Ions from different solutions (e.g., ESI samples such as reference compound 76, CE sample 78 and LC samples 80 & 82) are transferred from atmospheric pressure to a first differential pumping region 90 by gas flow via quadruple nozzle 84. Quadruple nozzle 84 comprises multiple sprayers at its exit end to eject ions from the different solutions in paths 86, 88, 92 & 94 aimed at an aperture in pressure restriction 98 (e.g., a skimmer), which transfers the ions from first pumping region 90 to second pumping region 96. An electric field applied across the exit end of quadruple nozzle 84 and restriction 98 as well as gas flow assist in the transfer of ions between these regions. Second differential pumping region 96 includes multipole 101 (comprising rods 102, 104, 106 & 108) which accepts ions of a selected mass/charge (m/z) ratio and guides them through second pressure restriction 100 and into TOF mass spectrometer 110.
Turning next to FIG. 4, shown is a prior art multiple needle electrospray apparatus for a mass spectrometer according to PCT Application No. PCT/CA99/00264 by applicant Synsorb Biotech, Inc., entitled “Electrospray Device For Mass Spectrometer” (“Synsorb”). As depicted, Synsorb's multiple needle electrospray apparatus includes a plurality of electrospray needles 120 mounted on a rotatable plate 112 for sequential injection of multiple sample streams. The rotatable electrospray apparatus allows collection of data from multiple sample streams by a single mass spectrometer 128 in a short time by rotating the electrospray apparatus to sequentially monitor the stream from each of the needles 120 for a brief duration before rotating the plate 112 to another of the needles.
According to one method for screening compound libraries which involve analysis of multiple sample streams by electrospray mass spectrometry, a compound library is prepared, such as by combinatorial chemistry techniques. Multiple sample streams each of which contain a compound library or sub-library are passed through a plurality of frontal chromatography columns. Each stream is passed through a single column to analyze the interaction of members of that sample stream with a target receptor within the column. The columns include a solid support or inert material on which the target receptor is bound or coupled. As the sample stream is continuously infused through the chromatography column. those compounds within the sample stream having a higher affinity for the target receptor (i.e., lipands) will be more strongly bound to the target receptors. When a compound has reached equilibrium with the column. it will break through and begin to pass out of the column with those compounds having the lowest affinity passing out of the column first. The sample streams exiting the chromatography columns are analyzed by electrospray mass spectrometry to determine the break through time for each compound. Mass spectrometry is particularly useful for this process because it allows for both detection and identification of the library members present in the sample streams exiting the columns.
FIG. 4 illustrates a prior art electrospray device for delivery of multiple liquid sample streams to a mass spectrometer according to Synsorb. The electrospray device includes electrospray chamber 114 for charging the droplets of a sample stream delivered by electrospray needles 120 and delivering the charged ions in a beam to mass spectrometer 128.
Electrospray needles 120 each have an upper end mounted on rotatable plate 112 in the circular arrangement. The lower ends of the electrospray needles may be rotated into a reproducible delivery position within electrospray chamber 114. The delivery position is at a precise location with respect to orifice 122 of mass spectrometer 128 which allows the sprayed droplets to be focused into a beam passing through orifice 122. The delivery position is within about ±0.5 mm of an ideal position in fluid connection with each of the electrospray needles 120 is a sample source such as chromatography columns 118 illustrated in FIG. 1. The chromatography columns 118 are mounted on the top of the rotatable plate 112 or are connected to the needles 120 with flexible lines.
Electrospray chamber 114 surrounds orifice 122 of the mass spectrometer and is open to atmospheric pressure, while surrounding needles 120 for containment purposes. Only needle 120 placed closest to a delivery position experiences a sufficiently high electric field and proximity for the efficient transmission of gas phase ions into the mass spectrometer 128. Further, electrospray needles 120 are coaxial needles which deliver the sample stream through an inner needle lumen and deliver a nebulizer gas, such as nitrogen, coaxially around the sample stream to break up the flow of the sample stream into a spray of droplets. Alternatively, the needles 120 may be single lumen needles delivering only the sample stream. The electrospray chamber 114 includes a charged sampling plate 116 surrounding the mass spectrometer entry orifice 122. The electrospray chamber 114 can also include an electrode 126 in the form of a half cylindrical member. The charged sampling plate 116 and half cylindrical electrode 126 are charged with an electric potential preferably of about 0 to 6000 volts. The electric field established by the sampling plate 116 and the electrode 126 surrounds the grounded needle 120 and imparts a charge to the sprayed droplets.
Alternatively, the charging of the sample stream droplets exiting electrospray needle 120 may be accomplished by use of a charged electrospray needle, biased sampling plate 116, and no electrode 126. The needle 120 may be continuously charged or may be charged only when the needle reaches the delivery position within electrospray chamber 114 by an electrical contact.
A counter current drying gas. such as nitrogen, is delivered to the electrospray chamber 14 through passageway 124 between charged sampling plate 116 and entry orifice 122 to assist in desolvating or evaporating the solvent from the sample stream to create fine droplets. Optionally, the drying gas may be delivered to electrospray chamber 114 in manners other than through passageway 124. In addition, the nebulizer gas may be delivered to the electrospray chamber 114 separately rather than by a co-axial flow through the electrospray needle. Both the nebulizer gas and the drying gas are introduced into the electrospray chamber 14 to obtain fine droplets of the sample stream. However, depending on the flow rate of the sample stream, the fine droplet size may be achieved without the need for a nebulizer gas and/or a drying gas.
The rotatable plate 112 is rotated by a motor connected to a drive shaft. The motor is interfaced with a controller to control the rotation of the plate and the dwell times for each of the needles.
During operation, multiple sample streams are continuously delivered to each of the chromatography columns 118 from sample sources by, for example, a pump, such as a syringe pump. The sample streams exiting columns 118 may be combined with a diluent in a mixing chamber or mixing tee 138 positioned between the column and needle 120. The sample streams pass continuously through electrospray needles 120 with a nebulizer gas delivered around the sample streams to break up the flow into droplets. In one disclosed embodiment, sample streams pass through all of the needles 120 simultaneously with only one of the streams from a needle positioned at the delivery position being analyzed by the mass spectrometer at a time. The sample streams from the remaining needles 120 are optionally collected by a tray 130 for delivery to waste.
To perform analysis of the multiple sample streams, Synsorb provides that rotatable plate 112 is stepped in one direction (e.g., counter clockwise), through approximately half of the needles 120. When a quadrupole mass spectrometer is used, a dwell time for each electrospray needle 120 ranges from about 0.5 to 10 seconds, preferably about 1 to 5 seconds before switching to the next column. After analysis of approximately half the sample streams, the rotatable plate 112 then returns clockwise to a home position and begins stepping in an opposite direction (e.g., clockwise), through the remaining half of needles 120. Finally, rotatable plate 112 returns again to the home position and repeats the procedure. The system operates continuously for a preset period of time related to the chromatographic requirements. Step times for rotation between successive needles is preferably less than about 100 msec, more preferably less than about 10 msec. The rotation of plate 112 in one direction followed by reversing the rotation is preferred to prevent the feed lines for feeding the sample streams from the pump to columns 118 from becoming twisted.
Alternatively, the sample source, the pump or alternative, and the feed lines for delivery of the sample streams to columns 118 may be mounted on plate 112. With this embodiment, plate 112 may be rotated continuously in one direction to sequentially analyze the flows from each of the needles without requiring the plate to reverse direction and return to a home position.
This multiple needle electrospray apparatus is described for use with any of the known mass spectrometers including a quadrupole mass spectrometer, quadrupole ion trap mass spectrometer, Penning or Paul ion trap mass spectrometer, FTICR (Fourier transform inductively coupled resonance) mass spectrometer, TOF mass spectrometer, and the like. A TOF mass spectrometer is preferred due to its high spectral acquisition rate (>100 spectra per second). However, the slower quadrupole mass spectrometer may also be used which can record spectra at a rate of approximately 0.5 to 1 per second. The dwell times for analysis of each sample stream will vary depending on the spectral acquisition of the mass spectrometer used.
Synsorb also discloses the use of different numbers of electrospray needles depending on the number of sample streams which are to be analyzed. The spacing of the multiple electrospray needles 120 is important to the operation of the electrospray device. In particular, electrospray needles 120 should be spaced sufficiently to prevent cross over effects resulting from the sample stream from one columns influencing the analysis of the sample stream of an adjacent column. In addition, electrospray needles 120 should be spaced as close together as possible to minimize the step times for rotation between adjacent needles. Preferably, the spacing between columns should be about 0.5 cm to 10 cm, depending on the mass spectrometer used. Alternatively, physical blocking members may be used to prevent cross over effects and allow closer needle placement.
Next, FIG. 5 shows a top view of another rotatable electrospray apparatus for delivery of sample streams to a mass spectrometer 140 according to Synsorb. The electrospray apparatus includes a plurality of electrospray needles 142 mounted in a radial arrangement on a rotatable plate 144. Each of the needles 142 are in fluid connection with a chromatography column 146. The radial arrangement of the electrospray needles 142 allows more columns 146 to be positioned on a rotatable plate 144 of a smaller diameter. According to this embodiment, the discharge ends of the needles 142 are preferably spaced a distance sufficient to prevent a cross over effect between adjacent needles. However, the columns 146 can be arranged close together around the periphery of the rotatable plate 144.
The present invention is distinguished from prior art by providing two distinct advantages. First, the preferred embodiment allows the use of heated drying gas and an endcap for efficient drying of sprayed droplets. Second, the sampling orifice of the multiple part capillary is, in the preferred embodiment, moved to an optimum position for the sampling of ions from a given sprayer, while in prior art designs, the sampling orifice was in a fixed position (not necessarily the optimum for any given sprayer). A result of this configuration (i.e., having a movable “sampling orifice”) is that the sampling orifice may be positioned closer to the sprayer, allowing use of a wider variety of spray devices, such as nanosprayers, microsprayers, which cannot be used with the prior art multiplexing devices.
The present invention further distinguishes itself from prior art by providing a means and method for simpler, more efficient, multiplexed sample introduction into an ESI mass spectrometer. According to prior art multiplexing apparatuses and methods, first, a sample spray is formed from the plurality of sprayers. Second, the device selects the specific sprayer from which to accept the sample spray. Third, the droplets from the sample spray are desolvated in an electric field wherein sample ions are formed. Fourth, the sample ions are transported into a mass spectrometer. This sequence of spraying, selecting, desolvating, and then transporting the sample ions has significant limitations and disadvantages. For example, the prior art multiplexing devices cannot be used adequately with nano- or micro-electrospray sources because the sampling orifice cannot be brought close enough to the sprayer(s). Also, the prior art cannot utilize different types of sprayers (i.e., electrospray, pneumatic spray, etc.) simultaneously. That is, electrospray (specifically, nanospray) cannot be used with drying gas while drying gas is needed for pneumatic sprayers. The prior art multiplexing designs do not function such that drying gas may be used with only some of the plurality of sprayers—it must be used with all or none. Further, in the prior art multiplexing devices, optimum conditions for maximum performance cannot be obtained for each sprayer independently—only a compromised arrangement may be obtained.
In contradistinction, the present invention uses a multiple section capillary device, which allows the orifice of the entrance to a mass analyzer to be moved (e.g., rotated) so as to sequentially sample ions from a series of ESI sprayers. The use of such an apparatus to multiplex samples from a plurality of sprayers necessarily provides a distinct and improved method of such sampling. Some of the distinct advantages provided by the present invention include use with nano- or micro-electrospray sources since the sampling orifice may be positioned at any distance from the sprayer(s) desired, the ability to simultaneously utilize any number of different types of sprayers (i.e., electrospray, pneumatic spray, etc.), and the ability to optimize the conditions for maximum performance and resolution for each sprayer, independently—a significant improvement over the prior art devices. Also, optionally, the use of an endcap electrode and drying gas in conjunction with a multiplexed sampling apparatus may be used to enhance the performance of an ESI/HPLC source for a mass spectrometer.